The proposed studies are based on our previous findings that in late gestation, basal plasma Cortisol levels are normal in the long-term hypoxic (LTH) sheep fetus, while expression of key adrenal steroidogenic enzymes (P450 cholesterol side chain cleavage [CYP11A] and P450 17a-hydroxylase [CYP17]) are suppressed. Paradoxically, basal plasma adrenocorticotropin (ACTH) concentrations are elevated and, in response to a secondary stressor, Cortisol production is enhanced compared to normoxic controls. Thus, it is apparent that the fetal HPA axis has acclimatized to LTH. However, the mechanisms driving this adaptation remain to be elucidated. Nitric oxide (NO) has profound inhibitory effects on steroidogenesis in a wide range of endocrine tissues and NO synthases (NOS) are subject to regulation by hypoxia. As such NO represents a potential mechanism in the adrenocortical adaptation to LTH. The proposed studies provide a logical extension of our previous observations on HPA function in the LTH fetus and examine specific mechanisms governing the adrenocortical adaptation to LTH. This proposal will test the general hypothesis that NO (generated by eNOS) mediates the adrenocortical adaptation to LTH preserving normal basal Cortisol production in the face of this chronic stressor, and that ACTH release in response to secondary, potentially life threatening, acute stressors overcomes NO inhibition of steroidogenesis. Specific studies will define the site(s) and mechanisms via which NO inhibits steroidogenesis in LTH ovine fetal adrenal cortical cells by determining the role of S-nitrosylation of key steroidogenic enzymes (CYP11A1, CYP17) and the major transcription factor governing CYP11A1 and CYP17 expression(SF-l). Additional studies are aimed at determining the mechanisms of LTH regulation of eNOS/protein interactions (Cav-1 and Hs90) and key signaling pathways governing eNOS activity in the ovine fetal adrenal (AMPK, PI3-K/Akt, MEK/ERK1/2). Further studies will determine the mechanism(s) by which elevated ACTH levels from a secondary stress-induced release of ACTH suppresses eNOS function and the potential role of eNOS in the regulation of expression of CYP17 and CYP11A1. The proposed studies are key to furthering our understanding ofthe mechanisms of fetal adaptations to LTH.